Structural elucidation of transmembrane transporter protein bilitranslocase: Conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy |
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Authors: | Amrita Roy Choudhury Andrej Perdih Špela Župerl Emilia Sikorska Tom Solmajer Stefan Jurga Igor Zhukov Marjana Novič |
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Institution: | 1. National Institute of Chemistry, Ljubljana, Slovenia;2. Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland;3. NanoBioMedical Centre, Adam Mickiewicz University, Poznan, Poland;4. Department of Macromolecular Physics, Faculty of Physics, Adam Mickiewicz University, Poznan, Poland;5. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland |
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Abstract: | Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of α-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two α-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds. |
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Keywords: | BTL bilitranslocase protein TM2 peptide representing the second (positions 73&ndash 95) transmembrane region of BTL protein TM2A peptide representing the second (positions 75&ndash 94) transmembrane segment of BTL protein TM2B peptide representing a prolonged second transmembrane segment (positions 73&ndash 99) of BTL protein TM3 peptide represented third transmembrane region (residues 220&ndash 237) of BTL protein HSQC Heteronuclear Single Quantum Correlation spectroscopy TAV time-averaged distance restraints RFD radial distribution function CD circular dichroism SDS sodium dodecyl sulfate DPC dodecylphosphocholine DPPC dipalmitoyl phosphatidyl choline DSS sodium 2 2-dimethyl-2-silapentane-5-sulfonate DLPC 1 2-dilauroyl-sn-glycero-3-phosphocholine DMPC 1 2-dimyristoyl-sn-glycero-3-phosphocholine DOPC 1 2-dioleoyl-sn-glycero-3-phosphocholine MD molecular dynamics CPNN counter-propagation neural network AMF atomic force microscopy FRET fluorescence resonance energy transfer SD steepest descent ABNR Adopted Basis Newton&ndash Raphson method |
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